Streptavidin (STA) and avidin proteins are homotetramers that have extraordinarily high binding affinities for the small molecule biotin (Kd−10−14 M)[1]. This remarkably strong and specific interaction between streptavidin or avidin and biotin has been exploited in a wide range of applications, such as bio-interfaces to nanostructure assembly and molecular labeling [2]. The tetrameric form of avidin proteins, however, may cause unwanted cross-linking of the biotin conjugates. For example, streptavidin-based labeling of biotinylated cell-surface receptors disrupted the normal function of the receptors through oligomerization [3]. However, the development of monomeric streptavidin or avidin without a dramatic decrease (Kd˜10−7-10−9 M) in biotin affinity has not been successful because a tryptophan residue from an adjacent subunit is critical for strong biotin binding [4-7]. Instead, a tetrameric but monovalent streptavidin with a single biotin binding site was developed [3]. Although this monovalent streptavidin is very effective for biotin labeling without cross-linking [8], a truly monomeric streptavidin or avidin protein is still highly desired for minimal perturbation of biotin-labeled targets and even more diverse applications of avidin proteins. For example, monomeric avidin can be genetically fused with other target proteins without oligomerization, and this fusion will also allow a new form of creation having different spatial organization and valency of the biotin binding sites.
Throughout the entire specification, many papers and patent documents are referenced and their citations are represented. The disclosure of the cited papers and patent documents are entirely incorporated by reference into the present specification and the level of the technical field within which the present invention falls, and the details of the present invention are explained more clearly.